SA is a pathogen, which causes diseases in virtually all mammalian species. SA is an important pathogen in the bovine, ovine and caprine species causing suffering and very high economical losses in the dairy breeds including cattle, sheep and goats (cattle) all over the world. The SA induced mastitis might be the single most important economical negative factor in veterinary medicine due to the pathogenicity including severe acute and painful inflammation, chronic and persistent conditions and the difficulties with efficient treatments mostly based on antibiotics that might not work because of the resistance leading culling. The alternative would be vaccination, but no efficient vaccine so far exists, which can constitute the basis for combating the mastitis problem caused by SA.
There are several pathogenicity factors for a prospective protective vaccine against SA infections to overcome. E.g. SA “hides” antigens in the cell essential for the infection and for its survival. Further SA has the capacity to manipulate the host immune system to facilitate the bacterial existents. This complex situation is one reason that an efficient SA vaccine for protection against mastitis is not available.
SA components that manipulate and may down regulate the immune response of the host that includes excreted products such as α- and β-toxins, leukocidin. Also cell bound components like super antigens and protein A is involved.
Examples of essential structures that the bacteria hide for the immune system of the host it infects is Fibronectin binding proteins (FnBp) or external fibrin factor (Ebf) essential for adherence of SA to tissue e.g. in wounds.
Several Fibronectin binding microbial surface components-recognizing adhesive matrixmolecules have been isolated and characterized from different Gram-positive bacteria. Genes encoding Fibronectin binding microbial surface components recognizing adhesive matrixmolecules from Staphylococcus aureus (Signas et al., “Nucleoside sequence of the gene for a fibronectin binding protein from Staphylococcus aureus: Use of this peptide sequence in synthesis of biologically active peptides,” Proc. Natl. Acad. Sci. USA. 86:699-173, 1989.), Streptococcus pyogenes (Talay et al., “Fibronectin-binding protein of Streptococcus pyogenes: Sequence of the binding domain involved in adherence of streptococci to epithelial cells,” Infect. Immun., 60:3837-3844, 1992.; Hansky et al., Infect. Immun., 60:5119-5125, 1992) and Streptococcus dysgalactiae (Lindgren et al., “Two different genes coding for fibronectin-binding proteins from Streptococcus dysgalactiae—the complete nucleotide sequences and characterization of the binding domains,” Eur. J. Biochem., 214:819-827, 1993.) have been cloned and sequenced. The deduced amino acid sequences revealed 60-100 kDa proteins with very similar structural organization. The N-terminal signal sequence is followed by a long stretch of unique sequence, which in some cases is interrupted by two copies of an approximately 30 amino acid long segment. The ligand binding site is located just N-terminal of a proline-rich domain, which is believed to anchor the proteins in the cell wall. This domain is followed by the sequence LPXTGX which is a cell wall targeting signal (Schneewind et al., Science, 268:103-106, 1995. et al., 1995), a stretch of hydrophobic residues representing a trans-membrane unit and a short C-terminal cytoplasmic domain containing a cluster of positively charged residues. Streptococcus agalactiae and Streptococcus uberis and coagulase negative Staphylococcus, have similar fibronectin binding devices and are of special interest for mastitis in the bovine and ovine species. Also coagulase negative Staphylococci have FnBp and are causing mastitis. The primary Fibronectin binding sites on these microbial surface components recognizing adhesive matrix molecules consist of 30-42 amino acid long motifs repeated 3-4 times, and most of the repeated units contain a consensus sequence (Lindgren et al., 1993 loc cit; McGavin et al., “Fibronectin receptors from Streptococcus dysgalactiae and Staphylococcus aureus: Involvement of conserved residues in ligand binding,” J. Biol. Chem. 268:23946-23953, 1993.). This domain is composed of a unit of 37-40 amino acids, repeated three or four times (FIG. 1 of U.S. Pat. No. 6,685,943).
Thus, FnBp is an important adhesion protein (antigen) for the formulation a protective vaccine against several gram-positive bacteria including SA based on the function of adhesion and even more as a target for phagocytosis being the main protective mechanism against SA. Importantly, FnBp is present in many isolates from gram-positive bacteria such as Streptococcus pyogenes and/or Streptococcus dysgalactiae and Streptococcus agalactiae and Streptococcus uberis and coagulase negative Staphylococcus, and coagulase negative Staphylococcus. Close to 100% of SA isolates. A vaccine would not only be for protection of cows against mastitis caused by SA infection, but also for virtually all animal species affected by infections caused by SA including man.
The composition of vaccine antigens might vary in vaccines against gram-positive bacteria, including SA infections and SA caused disease depending on several factors including local strains and clinical pictures. The adhesion to fibronectin mediates an important and common factor for the infection process, and this protein is present on virtually all SA isolates. The blocking of adhesion and neutralisation is an effect by IgG1 by antibodies, while IgG2a is important for phagocytosis the major immune protective mechanism against SA.
In view of the fact that SA also is an intracellular parasite the cell mediated arm of the immune system (CMI) is an essential factor. It is well documented that the iscom system potently enhances CMI and in particular the cytotoxic T cells killing infected cells e.g. SA infected cells. Thus, the adjuvant formulations based on the iscom-technology have the capacity to evoke several immune protective mechanisms.
Iscoms containing FnBp have been reported (Nickerson Nelson et al. 2000 (Symposium, Stresa Italy, Proceedings, 426-4319). A FnBp was incorporated into the iscom matrix to form an iscom with integrated antigen. Nelson's iscom formulation was however not sufficiently immunogenic and not useful for vaccines partly because of the short duration of immunity it induced. The present invention based on iscom matrixor liposomes as adjuvant gives a far better immunity (see present example 5 for comparison). Further, the vaccine formulation according to this invention is much better suited for large-scale production, since the iscom technology used in this invention only requires the addition of iscom matrix to the vaccine antigen suspension.
It has now turned out that the iscom technology is very well suited for preparation of vaccine compositions against gram-positive bacteria, including Staphylococcus aureus SA and especially against mastitis in the bovine species.